Characterization of Drosophila Germline Stem Cell Chromatin Using ChIP-Seq Summary. Our understanding of the genetic and molecular mechanisms that regulate the activity of human stem cells relies heavily on work using model organisms. Drosophila represents a powerful system for the study of diverse aspects of stem cell biology. Several complex regulatory networks governing stem cell behavior have been identified and characterized in the germline stem cells of the Drosophila ovary. One emerging theme across species is a role for chromatin-mediated gene regulation in stem cell maintenance. While a few individual genes that participate in establishing and regulating chromatin structure are known to be important for stem cell maintenance, a comprehensive view of the chromatin architecture in adult stem cells is lacking. Here we propose to purify ovarian germline stem cells (GSCs) and survey their chromatin landscape by genome-wide chromatin immunoprecipitation and massive parallel sequencing. We will examine the distribution of Polymerase II, as well as histone modifications associated with silence chromatin (e.g. trimethylation of histone H3 on lysine 27 (H3K27me3)) and active chromatin (e.g. trimethylation of histone H3 on lysine 4 (H3K4me3)) in purified GSCs. These studies will establish whether Drosophila stem cells contain bivalent chromatin domains, occupied by both H3K27me3 and H3K4me3, as well as genes occupied by poised polymerases, two features proposed to contribute to stem cell identity in mammalian cultured cells. The identification of chromatin features conserved across systems may reveal important mechanisms of gene regulation that are unique to stem cells. We will also examine GSCs as they differentiate, to describe how chromatin programming changes over developmental time. Our proposal to assay chromatin from highly purified stem cells isolated from adult tissues would be the first examination of the chromatin environment from stem cells in their native environment. These studies should reveal whether stem cells share unique chromatin features, and thus contribute to our understanding of the basic principles that govern stem cell self-renewal. PUBLIC HEALTH RELEVANCE: Regenerative medicine relies on an understanding of the basic principles that govern the self-renewing divisions of stem cells. This project aims to isolate pure stem cells from adult tissues and characterize the changes that occur in the DNA environment between self-renewing and differentiating stem cells.